PROJECT SUMMARY Membrane fusion, the merging of two membranes into a single continuous phospholipid bilayer, is central to intracellular trafficking, secretion, fertilization and other processes vital to living organisms. To fuse membranes, cells use a machinery whose core consists of the SNARE proteins. During exocytosis, neurotransmitters or hormones are released by neurons or endocrine cells, respectively, when synaptic vesicles or secretory granules fuse with the plasma membrane, driven by complexation of vesicular v-SNAREs with plasma membrane t- SNAREs that assemble into SNAREpin complexes. SNAREpin assembly pulls the membranes together and provokes their fusion. The result is a fusion pore which allows vesicle contents to be released through the plasma membrane. Exocytotic fusion pores, widely studied electrophysiologically, often flicker open and closed repeatedly before resealing (?kiss and run? fusion) or dilating (?full fusion?). The detailed mechanism of SNARE-mediated fusion is unknown. It is thought that once a fusion pore is created, SNAREs participate in regulating pore dynamics and openness post-fusion, thereby regulating the amount and rate of contents released. However, the mechanisms underlying this regulation are not known. A major obstacle to answering these questions has been the lack of quantitative modeling approaches that can access physiologically relevant fusion timescales (msec-sec). Atomistic and current coarse-grained molecular dynamics (MD) simulation approaches yield vital information, but due to computational limitations cannot describe long time collective fusion phenomena. We will develop two coarse-grained methods to access the necessary timescales. One is a coarse-grained continuum approach, with bilayers represented as continuous fluctuating deformable surfaces; the other a more detailed MD simulation adapting an existent simulation with highly coarse-grained explicit phospholipids. A multiscale strategy is proposed: both methods coarse-grain the SNAREs, but dimensions, surface charge, zippering energy landscape and other features will be described by realistic parameters from experiment or less coarse grained simulations. These methods will simulate many SNAREpins at the pre-fusion site to assess if they cooperatively fuse membranes, and to map the network of pathways to fusion that may involve hemifused or extended contact intermediate states. We will then simulate the dynamical fusion pore itself and study for the first time how the forces from assembled SNAREpins affect the flickering dynamics and dilation of the pore. Once working simulations of multiple SNAREpins operating between dynamic membranes are in place, we will progressively ?reconstitute? the fusion machinery with successive layers of complexity, adding the SNARE regulating proteins complexin and synaptotagmin to test candidate mechanisms whereby these components clamp or activate SNARE-mediated fusion. These enlarged models will be used to build molecularly explicit models of Ca2+-regulated neurotransmitter release at synapses.